Assessment of insecticidal effects and selectivity of CAPA-PK peptide analogues against the peach-potato aphid and four beneficial insects following topical exposure.

BACKGROUND: Insect Capability neuropeptides (CAP2b/CAPA-PKs) play a critical role in modulating different physiologies and behavior in insects. In a previous proof-of-concept study, the CAP2b analogues 1895 (2Abf-Suc-FGPRLamide) and 2129 (2Abf-Suc-ATPRIamide) were reported to reduce aphid fitness when administered by injection. In the current study, the insecticidal efficacy of 1895 and 2129 on the peach potato aphid Myzus persicae was analyzed by topical application, simulating a spray application scenario in the field. Additionally, the selectivity of the tested analogues was evaluated against a selection of beneficial insects, namely three natural enemies (Adalia bipunctata, Chrysoperla carnea and Nasonia vitripennis) and a pollinator (Bombus terrestris). RESULTS: Within 3-5 days post topical exposure of aphids to 1895, higher mortality (33%) was observed, as was the case for the treatment with 2129 (17%) and the mixture of 1895 + 2129 (47%) compared to the control (3%). 1895 and the mix 1895 + 2129 showed the strongest and comparable insecticidal effects. Additionally, surviving aphids treated with 1895 showed a reduction in total lifetime reproduction (GRR) of 30%, 19% with 2129 and 39% with the mix 1895 + 2129. Of interest from a biosafety perspective is that by using the same delivery method and dose, no significant effects on survival, weight increase and food intake was observed for the representative natural enemies and the pollinator. CONCLUSION: This study highlights the potential of exploiting CAP2b analogues such as 1895 (core structure FGPRL) as aphicides. Additionally, the CAP2b analogues used in this study were selective as they showed no effects when applied on four representative beneficial insects.

An ultraviolet B condition that affects growth and defense in Arabidopsis.

Ultraviolet B light (UV-B, 280-315 nm) is the shortest wavelength of the solar spectrum reaching the surface of the Earth. It has profound effects on plants, ranging from growth regulation to severe metabolic changes. Low level UV-B mainly causes photomorphogenic effects while higher levels can induce stress, yet these effects tend to overlap. Here we identified a condition that allows growth reduction without obvious detrimental stress in wild type Arabidopsis rosette plants. This condition was used to study the effects of a daily UV-B dose on plant characteristics of UV-B adapted plants in detail. Exploration of the transcriptome of developing leaves indicated downregulation of genes involved in stomata formation by UV-B, while at the same time genes involved in photoprotective pigment biosynthesis were upregulated. These findings correspond with a decreased stomatal density and increased UV-B absorbing pigments. Gene ontology analysis revealed upregulation of defense related genes and meta-analysis showed substantial overlap of the UV-B regulated transcriptome with transcriptomes of salicylate and jasmonate treated as well as herbivore exposed plants. Feeding experiments showed that caterpillars of Spodoptera littoralis are directly affected by UV-B, while performance of the aphid Myzus persicae is diminished by a plant mediated process.

High entomotoxicity and mechanism of the fungal GalNAc/Gal-specific Rhizoctonia solani lectin in pest insects.

Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cotton leaf-worm Spodoptera littoralis and the pea aphid Acyrthosiphon pisum demonstrated a high concentration-dependent entomotoxicity, suggesting that this GalNAc/Gal-specific fungal lectin might be a good control agent for different pest insects. RSA at 10 mg/g in the solid diet of 2nd-instar caterpillars caused 84% weight reduction after 8 days with none of the caterpillars reaching the 4th-instar stage. In sucking aphids, 50% mortality was achieved after 3 days with 9 µM of RSA in the liquid diet. Feeding of FITC-labeled RSA to both insect pest species revealed strong lectin binding at the apical/luminal side of the midgut epithelium with the brush border zone, suggesting the insect midgut as a primary insecticide target tissue for RSA. This was also confirmed with cell cultures in vitro, where there was high fluorescence binding at the microvillar zone with primary cultures of larval midgut columnar cells of S. littoralis, and also at the surface with the insect midgut CF-203 cell line without lectin uptake in the midgut cells. In vitro assays using insect midgut CF-203 cells, revealed that RSA was highly toxic with an EC50 of 0.3 µM. Preincubation with GalNAc and saponin indicated that this action of RSA was carbohydrate-binding dependent and happened at the surface of the cells. Intoxicated CF-203 cells showed symptoms of apoptosis as nuclear condensation and DNA fragmentation, and this concurred with an increase of caspase-3/7, -8 and -9 activities. Finally, RSA affinity chromatography of membrane extracts of CF-203 cells followed by LC-MS/MS allowed the identification of 5747 unique peptides, among which four putatively glycosylated membrane proteins that are associated with apoptosis induction, namely Fas-associated factor, Apoptosis-linked gene-2, Neuroglian and CG2076, as potential binding targets for RSA. These data are discussed in relation to the physiological effects of RSA.

GalNAc/Gal-binding Rhizoctonia solani agglutinin has antiproliferative activity in Drosophila melanogaster S2 cells via MAPK and JAK/STAT signaling.

Rhizoctonia solani agglutinin, further referred to as RSA, is a lectin isolated from the plant pathogenic fungus Rhizoctonia solani. Previously, we reported a high entomotoxic activity of RSA towards the cotton leafworm Spodoptera littoralis. To better understand the mechanism of action of RSA, Drosophila melanogaster Schneider S2 cells were treated with different concentrations of the lectin and FITC-labeled RSA binding was examined using confocal fluorescence microscopy. RSA has antiproliferative activity with a median effect concentration (EC(50)) of 0.35 µM. In addition, the lectin was typically bound to the cell surface but not internalized. In contrast, the N-acetylglucosamine-binding lectin WGA and the galactose-binding lectin PNA, which were both also inhibitory for S2 cell proliferation, were internalized whereas the mannose-binding lectin GNA did not show any activity on these cells, although it was internalized. Extracted DNA and nuclei from S2 cells treated with RSA were not different from untreated cells, confirming inhibition of proliferation without apoptosis. Pre-incubation of RSA with N-acetylgalactosamine clearly inhibited the antiproliferative activity by RSA in S2 cells, demonstrating the importance of carbohydrate binding. Similarly, the use of MEK and JAK inhibitors reduced the activity of RSA. Finally, RSA affinity chromatography of membrane proteins from S2 cells allowed the identification of several cell surface receptors involved in both signaling transduction pathways.

Biostable and PEG polymer-conjugated insect pyrokinin analogs demonstrate antifeedant activity and induce high mortality in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae).

The pyrokinins (PK) are multifunctional neuropeptides found in a variety of arthropod species, including the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae). A series of biostable pyrokinin analogs based on the shared C-terminal pentapeptide core region were fed in solutions of artificial diet to the pea aphid over a period of three days and evaluated for antifeedant and aphicidal activity. The analogs contained either modified Pro residues Oic or Hyp and or a d-amino acid in key positions to enhance resistance to tissue-bound peptidases and retain activity in a number of PK bioassays. A series of PK analogs conjugated with two lengths of polyethyleneglycol (PEG) polymers were also evaluated in the aphid feeding assay. Three of the biostable PK analogs demonstrated potent antifeedant activity, with a marked reduction in honeydew formation and very high mortality after 1 day. In contrast, a number of unmodified, natural pyrokinins and several other analogs containing some of the same structural components that promote biostability were inactive. Two of the most active analogs, Oic analog PK-Oic-1 (FT[Oic]RL-NH(2)) and PEGylated analog PK-dF-PEG(8) [(P(8))-YF[dF]PRL-NH(2)], featured aphicidal activity calculated at LC(50)'s of 0.042nmol/µl [0.029µg/µl] (LT(50) of 1.0 day) and 0.126nmol/µl (LT(50) of 1.3 days), respectively, matching the potency of some commercially available aphicides. Notably, a PEGylated analog of a PK antagonist can block over 55% of the aphicidal effects of the potent PK agonist PK-Oic-1, suggesting that the aphicidal effects are mediated by a PK receptor. The mechanism of this activity has yet to be established, though the aphicidal activity of the biostable analogs may result from disruption of digestive processes by interfering with gut motility patterns, a process shown to be regulated by the PKs in other insects. The active PK analogs represent potential leads in the development of selective, environmentally friendly aphid pest control agents.

Insecticidal properties of Sclerotinia sclerotiorum agglutinin and its interaction with insect tissues and cells.

This project studied in detail the insecticidal activity of a fungal lectin from the sclerotes of Sclerotinia sclerotiorum, referred to as S. sclerotiorum agglutinin or SSA. Feeding assays with the pea aphid (Acyrthosiphon pisum) on an artificial diet containing different concentrations of SSA demonstrated a high mortality caused by this fungal lectin with a median insect toxicity value (LC50) of 66 (49-88) µg/ml. In an attempt to unravel the mode of action of SSA the binding and interaction of the lectin with insect tissues and cells were investigated. Histofluorescence studies on sections from aphids fed on an artificial liquid diet containing FITC-labeled SSA, indicated the insect midgut with its brush border zone as the primary target for SSA. In addition, exposure of insect midgut CF-203 cells to 25 µg/ml SSA resulted in a total loss of cell viability, the median cell toxicity value (EC50) being 4.0 (2.4-6.7) µg/ml. Interestingly, cell death was accompanied with DNA fragmentation, but the effect was caspase-3 independent. Analyses using fluorescence confocal microscopy demonstrated that FITC-labeled SSA was not internalized in the insect midgut cells, but bound to the cell surface. Prior incubation of the cells with saponin to achieve a higher cell membrane permeation resulted in an increased internalization of SSA in the insect midgut cells, but no increase in cell toxicity. Furthermore, since the toxicity of SSA for CF-203 cells was significantly reduced when SSA was incubated with GalNAc and asialomucin prior to treatment of the cells, the data of this project provide strong evidence that SSA binds with specific carbohydrate moieties on the cell membrane proteins to start a signaling transduction cascade leading to death of the midgut epithelial cells, which in turn results in insect mortality. The potential use of SSA in insect control is discussed.

Glycosylation signatures in Drosophila: fishing with lectins.

Glycosylation is a co- and/or post-translational protein modification that generates enormous structural diversity among glycoproteins. In this study, immobilized lectins were used to capture glycoproteins with different glycan profiles from Drosophila melanogaster extracts. On the basis of previous results from glycan array analyses, the snowdrop (Galanthus nivalis) agglutinin (GNA), the tobacco (Nicotiana tabacum) lectin (Nictaba) and the Rhizoctoni solani agglutinin (RSA) were used to select for a broad range of N- and O-glycan structures. After different lectin affinity chromatographies, the glycoproteome of Drosophila was analyzed using LC-MS/MS and glycoprotein abundances were calculated by different label-free methods. Bioinformatics tools were used to annotate the identified glycoproteins and the glycoproteins were classified according to their molecular function or their involvement in a biological process. Subsequent enrichment analysis (using the DAVID database) was employed to find biological processes or molecular functions in Drosophila in which a particular glycan signature is overrepresented. The results presented here clearly demonstrate that next to the presence of high-mannose and pauci-mannose N-glycans, Drosophila is capable of synthesizing glycoproteins carrying extended hybrid and complex N-linked glycans. Furthermore, it was demonstrated that a specific glycosylation signature can be associated with a functionally related group of glycoproteins in Drosophila, both in terms of biological process and molecular function.